microlab

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Author:
narasha
ID:
109867
Filename:
microlab
Updated:
2011-11-29 00:06:41
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micro
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micro
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  1. Aseptic Technique
    • insures that no contaiminating organisms are introduced into culture materials
    • insures that organisms being handled do not contaminate the handler or others
    • no contamination remains in the work area after you have worked there
  2. Aseptic TECHNIQUES
    • disinfect work area before and after
    • sterilize loops and needles in flame before and after
    • culture tube flaming before and after (never lay cap on table)
  3. Wet mount
    • material suspended in a solution usually DI water
    • at end, place coverslip over water at 45 degree angle
  4. Fixed Slide
    • material is tightly adhered to slide - usually dead
    • air dry
    • run over heat
  5. Heat Fixation Purpose and Benefits ***
    • adheres the material to the slide
    • kills any living bacteria
    • preserves the shape of the cells
  6. Why Stain?
    • Microbial cells lack contrast
    • so to see them a dye must be used to stain them
  7. Basic Stains & examples
    • cationic (positive charge)
    • Ex: methylene blue
    • crystal violet
    • safranin
    • fuchsin
    • Most bacteria readily stain with basic dyes
  8. Acidic Stains & Examples
    • anionic (negative charge)
    • Eosin
    • Nigrosin
    • Indian Ink
  9. Simple
    Differential
    Structural
    • Simple: 1 dye
    • Differential: primary and counterstain
    • Structural: used to identify cell parts not revealed by conventional staining (ex. spores)
  10. Gram Stain
    • 125 year old method
    • Hans Gram
    • used to find ID of bacteria
  11. Gram Positve
    Gram Negative
    • Purple
    • pink
  12. Bacillus only divide in ___ plane
    1
  13. Circle shape...
    coccus
  14. Cocci divide in....
    1 plane
    2 plane
    3 plane
    random planes
    • diplo or strepto
    • tetrad
    • sarcina
    • staphylo
  15. Spirillum
    spirochete
    • thick rigid spiral
    • a thin flexible spiral
  16. Simple Staining
    • 1% crystal violet used
    • one minute rinse
  17. Gram Staining Process**
    • Smear
    • Air dry
    • heat fix
    • Gram's crystal violet
    • 1 minute
    • rinse
    • Grams Iodine
    • 1 minute
    • rinse
    • 95% ethyl alcohol
    • rinse
    • Gram's Safranin
    • 1 minute
    • rinse
    • bibulous paper
  18. Negative Staining Process
    • One edge of the slide put a drop of nigrosin, the species
    • spread across slide using another slide
    • air dry
    • microscope it!!
  19. ***The 5 I's of Culturing Microbes***
    • Inculation
    • Incubation
    • Isolation
    • Inspection
    • Identification
  20. Inoculation
    introduction of a sample into a container of sterile media to produce a culture of observable growth
  21. Incubation
    under conditions that allow the organism to grow
  22. Isolation
    separating one species form another
  23. Inspection
    • macroscopically for color, texture, size, gas production
    • microscopically for cell shape, size and motility
  24. Identification
    staining,biochemical for metabolism, immunological or genetic
  25. Basic requirements for bacteria
    • carbon source,
    • nitrogen,
    • vitamins,
    • growth factors,
    • water
  26. Media can be classified according to 3 properties:
    • physical state
    • chemical composition
    • Functional type
  27. Physical States of Media
    explain each
    • Liquid: broth
    • Semisolid: clot-like consistency, contains some solidifying agent (agar or gelatin)
    • Solid: firm surface for colony formation; contains solidifying agent,
  28. Chemical Composition of Media
    • Synthetic: contains pure organic and inorganic compounds
    • Complex or non synthetic: contains at least one ingredient that is not chemically definable
  29. Functional Type of Media
    explain each
    • General: broad range of microbes
    • Enriched Media: contain complex organic substance
    • Selective Media: contains one or more agents that inhibit the growth fo some microbes and encourgae the growth of others
    • Differential: allows growth of several types of microbes and display visible differences among desired and undesired microbes
  30. Most commonly used Media
    • Nutrient broth: liquid medium; beef extract, peptone
    • nutrient agar: solid media; beef extract, peptone and agar
  31. Key Words:
    Enriched
    Selective
    Differential
    • Enriched: fastidious; milk, blood, brain, heart
    • Differential: determine, distinguish, detect, identify
    • Selective: inhibits, restricts, isolates
  32. Reducing Medium
    contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria
  33. Carbohydrate Fermentation Medium
    • contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction
    • basis for identifying bacteria and fungi
  34. Transport Media
    contain buffers and absorbants to prevent cell death but will not promote growth
  35. Assay Media
    to test drug, antiseptic, cosmetic, preservtive or disinfectant effect on microbes
  36. Enumeration Media
    used in industry and enviromental studies to count microbes in milk, water, soil, etc
  37. Agar & Gelatin
    • Agar: solid @ room temp; has carbon in it
    • Gelatin:colage form animal skin and bones; proteins (amino acids); liquid/semi-solid @ room temp
  38. Obligate aerobes & anaerobes
    Facultative Anaerobes
    Microaerophiles
    Aerotolerant
    • require oxygen
    • cannot stand oxygen
    • use oxygen if present but can grow without
    • live in enviro. where oxygen level is low
    • can live in oxygen but dont use it
  39. pH
    too high?
    too low?
    • most media are neutral range
    • HCl
    • NaOH
  40. Methods of Sterilization
    • Heat; autoclave
    • filtration: millipore filter
    • chemicals
  41. Colony Characteristics
    • punctiform: small dots
    • circular: perfect circles
    • filamentous: hair like projections
    • Irregular: majority of colonies
  42. Margin
    Colony characteristics
    • entire: circular colony
    • undulate: jagged edge
    • lobate: long finger like projections(may see a few)
    • Filamentous: hair like (prob. wont see)
    • Curled and Erose (irregularly notched)
  43. Elevation
    Colony Characteristics
    • flat
    • raised
    • convex (higher center)
    • pulvinate (much higher center)
    • umbonate (flat outer edge with bump in center)
    • Concave (higher edge than center)
  44. Texture
    Colony Characteristics
    dull, shiny, dry, gooey, slimy, smooth, crumbly
  45. Growth Characteristics in Liquid Media
    • Uniform: small particles
    • Floccular: large particles
    • Sedimentary: layer at bottom
    • Pellicle: layer on top
  46. Isolating Techniques
    • Streak Plate Technique
    • Pour plate TEchnique
    • Spread Plate Technique
  47. Starch Agar
    media type
    • Starch hydolysis
    • flood with iodine
    • dark blue: starch present: negative
    • no dark blue: starch gone: positive
  48. enriched
  49. Nutrient Gelatin Stab
    media type
    • medium solid after 15: negative
    • medium liquid after 15: positive
  50. differential
  51. Glucose Broth, Lactose, Sucrose
    media type
    • yellow: acid
    • blue: base: breakdown proteins not sugar
    • gas production
    • Carbohydrate fermenting media
  52. Kligler Iron Agar
    media type
    • hydrogen sulfide production
    • black deposit +
    • enriched, selective, differential
  53. Tryptone
    media type
    • indole production
    • +: red ring after addtion of Kovac
    • -: no red ring
    • enriched, differential
  54. Nitrate Broth
    media type
    • reduction of nitrate to nitrites
    • +: bubbles with nitrite solution
    • -: no bub bubs
  55. Nutrient Agar
    media type
    • Catalase Test
    • +: foaming with perioxide
    • -: no foaming
    • general purpose media
  56. Two Levels of Bacterial Growth
    • A cell increases in size
    • # of cells increases
  57. Generation
    the period between a bacteria cell beginning and the time of producing offspring
  58. Stages of Bacteria Growth
    • Lag phase
    • Exponential
    • Stationary
    • Death
  59. Methods of Counting Bacteria
    • Direct Methods
    • Most Probable Number (MPN)
    • Standard Plate Count (SPC)
    • Indirect Methods ex: spectrophotometer
  60. Strict Aerobes (obligate aerobes)
    Plenty of oxygen
  61. Strict Anaerobes (obligate anaerobes)
    No oxygen
  62. Facultative Anaerobe
    • Grow best in air
    • growth throughout entire test tube, but heavier on surface of agar
  63. Microaerophilic
    reduction of oxygen
  64. Aerotolerant (Indifferent)
    Oxygen Don't matter
  65. Spectrophotometer
    • Curve is set up
    • blank with nutrient broth
    • measure OD reading of sample
    • Calculate populations against established chart
  66. Calculation of Colonies after Dilution
    # of colonies counted x dilution factor of plate = number of CFU/mL
  67. CFU
    colony forming units
  68. **Know tube dilutions**
  69. Bacteria with a morphology of Bacillus
    • bacillus cereus
    • bacillus megaterium
    • enterobacter aerogenes
    • escherichia coli
    • serratia marcescens
  70. bacteris with a morphology of coccus
    • Micrococus luteus
    • microccus roseus
    • sarcina lutea
    • Staphylococcus epidermis
  71. Gram negative Bacteria
    • e. aero
    • e. coli
    • staph. epidermidis
  72. Gram positive bacteria
    • b. cereus
    • m. luteus
    • m. roseus
    • s. lutea
    • staph. epi
    • s. marcescens
  73. Only bacteria positive for hydrogen sulfide production
    Sarcina lutea
  74. Two bacterias positive for starch
    • b. cereus
    • b. megaterium
  75. Bacteria Negative for Gelatin Hydrolysis
    • E.aero
    • e. coli
    • m. roseus
  76. Bacteria positive for indole reduction
    • e.coli
    • sarcina lutea
  77. Negative for Nitrate reduction
    • micrococcus luteus
    • bacillus megaterium

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